Human Saliva Induces Acetoin-Dependent Metabolic Adaptation that Promotes Stress Tolerance in Streptococcus mutans

Abstract

Oral microbes are continually exposed to host-derived saliva, yet its effects on bacterial metabolism and stress adaptation remain incompletely defined. We investigated how saliva supplementation alters the physiology of the caries-associated bacterium Streptococcus mutans using transcriptomics (RNA-Seq), targeted metabolite measurements, specific gene deletions, and transposon sequencing (Tn-Seq). Growth in a chemically defined medium supplemented with pooled saliva altered expression of genes involved in carbohydrate transport, purine biosynthesis, and pyruvate-associated metabolism, including induction of the acetoin dehydrogenase operon. Saliva significantly increased extracellular acetoin accumulation across all S. mutans isolates tested, whereas little accumulation occurred in two commensal streptococcal species examined. Deletion of acetolactate synthase/decarboxylase (alsSaldB) abolished the saliva-associated increase, while deletion of individual acetoin dehydrogenase components (adhC / lplA) enhanced accumulation, supporting the predicted roles of genes in acetoin synthesis and catabolism. Increasing the available carbohydrate concentrations within the media further increased acetoin accumulation, alongside declining terminal culture pH that was significantly lower in cultures supplemented with saliva. Following growth with saliva, S. mutans exhibited approximately 10,000-fold greater survival during a hydrogen peroxide challenge than cells grown without saliva. Mutants within the acetoin pathway retained saliva-associated protection but showed approximately 100-fold lower survival than the wild type. Serial passage of a pooled transposon library identified previously unrecognized saliva-associated fitness determinants, including depletion of mutants carrying insertions in the acetoin dehydrogenase operon. Together, these findings identify acetoin metabolism as a contributor to saliva-associated metabolic remodeling and oxidative stress tolerance in S. mutans and establish saliva as a critical modulator of S. mutans physiology.